Alpha-2-macroglobulin (A2M) and related proteins share the function of acting as a molecular trap. Various types of compounds, such as hormones, growth factors, cytokines and proteins, can be “trapped” by A2M. A2M can bind both host and foreign peptides and particles. A2M is sometimes referred to as a panproteinase inhibitor since it can interact with a wide variety of proteinases. When captured by A2M, a proteinase is protected from large proteinase inhibitors and large substrates, but it can still interact with small inhibitors and substrates.
One A2M-proteinase association that has been well studied is the trapping of thrombin in A2M.
Thrombin is a pivotal enzyme in the coagulation cascade (2, 3) since it can, among other things, activate fibrinogen to form fibrin (4, 5), feedback to activate factors XI, VIII and V higher up in the coagulation cascade and activate platelets (2). Control of thrombin occurs, in part, via inhibition from the plasma proteins antithrombin (AT), heparin cofactor II and A2M. Reaction with AT is often the major inhibitory mechanism for control of thrombin activity in adults. However, in newborns and children, reaction with A2M is considered significant due to the elevated plasma concentrations of A2M compared to those of adults (6, 7, 8). Thrombin activation is hindered by A2M via cleavage of the A2M polypeptide chain, resulting in a conformational change in the 3-dimensional A2M structure to form a product in which the thrombin is captured within the A2M molecule (9, 10, 11). Within this thrombin-A2M complex, thrombin may also become covalently linked to the A2M through further reaction of thrombin amino groups with a thiol-lactone group on A2M (9, 12).
Given the importance of thrombin in the coagulation system, it is becoming increasingly desirable to develop a system to measure the thrombin generating potential in plasma or blood from patients to assess their haemostatic status. Previous methodology for measuring thrombin generation has either involved subsampling of activated plasma to detect thrombin activity (13, 14, 15) or detection of thrombin continuously using substrate mixed into the thrombin generating activated plasma (16, 17, 18). A major drawback of previous procedures for determining thrombin generation is that the reagents used to detect thrombin activity detect thrombin bound to A2M as well as thrombin not bound to A2M. In the case of subsampling methods, activity in assays of thrombin-A2M reaction with substrate (detected after neutralization of free unbound thrombin with added AT+heparin) are subtracted from assays for total thrombin activity to obtain free physiologically active thrombin concentrations. However, these experiments are laborious and require two thrombin generation assays to be carried out on each plasma sample. In addition, subsampling analyses cannot be done on coagulating plasma or blood. Alternatively, continuous measurement of thrombin generation using thrombin substrates added to the activated thrombin generating system (plasma, blood, etc) are unable to directly measure generation of thrombin that is not bound to A2M. In the continuous assays, theoretical amounts of physiologically active thrombin are determined by subtracting a calculated hypothetical curve of thrombin-A2M generation from the measured thrombin+thrombin-A2M data.
Various attempts have been made to develop assays that can measure the thrombin potential in plasma and blood. For example, U.S. Pat. No. 5,192,689 to Hemker et al. is directed to a method for determining the endogenous thrombin potential of plasma and blood. However, the patented method measures total thrombin activity, which includes thrombin alone as well as thrombin bound to A2M. The thrombin activity is measured using small chromogenic/fluorogenic substrates, followed by a calculated estimate of thrombin bound to A2M which is then subtracted from the total thrombin activity to give an estimated number for the free physiologically active thrombin.
Australian Patent Application No. 2003248589 is directed to another method for measuring thrombin potential. However, this method also includes a subtraction of the thrombin-A2M complex, detected by small substrates, from the total thrombin activity.
United States Patent Application Publication No. US 2005/0221414 is directed to a kit for measuring the thrombin generation potential in a sample of a patient's blood or plasma. In the kit described in this publication, all of the reagents such as the activator, substrate, CaCl2, etc. are in a dry mixture in the container waiting for a plasma or blood to be added for assay. Again, however, this kit would measure both free thrombin and thrombin bound to A2M.
U.S. Pat. No. 6,740,496 to Hemker describes a sterically hindered substrate system that has reduced reactivity against protease-A2M complexes compared to the free proteolytic enzyme. However, the claimed substrate invention is entirely restricted to a water soluble substrate molecule with a minimum size of 10 kDa. Free thrombin determination by the soluble hindered substrate is limited in clotting plasma or blood due to a number of issues, such as: problematic detection of the reporter molecule due to quenching by protein or cells, potential absorption or aggregation of the substrate on cells or polymerizing fibrin, and inability of the substrate to react with free thrombin itself resulting from surface hindrance of extremely large molecules.
In spite of many advances in the field, there remained an unmet need for an assay that can measure free thrombin as opposed to measuring both free thrombin and thrombin bound to A2M. The present invention addresses the need for such a methodology.